Journal of Agricultural Technology
Anti-bacterial activity of Caesalpinia coriaria (Jacq.) Willd. against plant pathogenic Xanthomonas pathovars: an ecofriendly approach
D.C. Mohana and K.A. Raveesha*. Agricultural Microbiology Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore. India. Mohana, D.C. and Raveesha, K.A. (2006). Anti-bacterial activity of Caesalpinia coriaria (Jacq.) Willd. against plant pathogenic Xanthomonas pathovars: an eco-friendly approach. Journal of Agricultural Technology 2(2): 317-327. Powdered leaf and pod material of Caesalpinia coriaria (Jacq.) Willd. was extracted with water and successively with different solvents viz., petroleum ether, benzene, chloroform, methanol and ethanol. Anti-bacterial activity assays of all the extracts against the important phytopathogenic Xanthomonas pathovars, known to cause diseases in tomato, french bean and cotton, were carried out by cup diffusion method. Aqueous pod extract showed significant activity. Among the five solvents extracts tested, methanol extract of both leaf and pod was most active against all the test bacteria, followed by ethanol extract. Comparison of the inhibitory activity of the extracts with the antibiotics bacterimycin 2000 and streptocycline revealed that methanol and ethanol extract of both leaf and pod and aqueous extract of pod were significantly higher than that of the antibiotics tested. Phytochemical analysis of leaf and pod materials revealed that antibacterial activity is due to the presence of phenolic and acidic fraction. Further separation of active fraction resulted in the loss of anti-bacterial activity, indicating a synergistic effect of the isolated active fraction. The results suggest that C. coriaria is a potential candidate plant for the management of phytopathogenic Xanthomonas which are known to cause diseases on cotton, french beans and tomato. Keywords: anti-bacterial activity, Caesalpinia coriaria, Xanthomonas pathovars
Introduction Pesticides are an essential input for preventing pre and post harvest crop losses (Mathur and Tannan, 1998; Saksena, 2001; Wheeler, 2002). Synthetic pesticides are commonly used to control phytopathogenic microorganisms (Agrios, 1997). Incessant and extensive use of these synthetic pesticides are posing serious problem to the life supporting systems due to their residual toxicity (Ferrer and Cabral, 1991; Gassner et al., 1997; Andrea et al., 2000; *
Corresponding author: Raveesha, K.A.; e-mail:
[email protected] 317
Harris et al., 2001; Campos et al., 2005). It is estimated that hardly 0.1% of the agro-chemicals used in crop protection reaches the target pest, leaving the remaining 99.9% to the environment to cause hazards to non target organisms including humans (Pimentel and Levitan, 1986). The large numbers of synthetic pesticides have been banned in the western world because of their undesirable attributes such as high and acute toxicity, long degradation periods, accumulation in the food chain and an extension of their power to destroy both useful and harmful pests (Barnard et al., 1997; Wodageneh and Wulp, 1997; Ortelli et al., 2005). In-spite of use of all available means of plant protection, about 1/3 of the yearly harvest of the world is destroyed by pests and loss due to this is expected to be nearly $300 billion per year (Chandler, 2005). Many phytopathogenic bacteria have acquired resistance to synthetic pesticides (Sundin et al., 1994; Clarke et al., 1997; Williams and Heymann, 1998; White et al., 2002). Pathovars of Xanthomonas are known to cause diseases on several vegetable and cash crop and are reported to have developed resistance to kanamycin, ampicillin, penicillin and streptomycin (Weller and Saettler, 1980; Nafade and Verma, 1985; Verma et al., 1989; Bender et al., 1990; Rodriguez et al., 1997). This seriously hinders the management of diseases of crops and agricultural products (Dekker, 1987). Considering the deleterious effects of synthetic pesticides on life supporting systems, there is an urgent need to search for alternative approaches for the management of plant pathogenic microorganisms (Hostettmann and Wolfender, 1997). Green plants represent a reservoir of effective chemotherapeutants and can provide valuable sources of natural pesticides (Balandrin et al., 1985; Mahajan and Das, 2003). Biopesticides has been suggested as an effective substitute for chemicals (Verma and Dubey, 1999; Kapoor, 2001). Reports are available on the use of several plant by-products, which posses antimicrobial properties, on several pathogenic bacteria and fungi (Dorman and Deans, 2000; Parameswari and Latha, 2001; Rath et al., 2001; Britto and Senthilkumar, 2001; Bylka et al., 2004; Shimpi and Bendre, 2005; Kilani, 2006), but reports are not available on the evaluation of inhibitory action of plants extract on phytopathogenic bacteria particularly in different pathovars of Xanthomonas which are known to cause many diseases in a wide variety of crops, causing considerable losses in yield and quality. This led the authors to screen in vitro, a large number of plants for antibacterial activity against important seed borne phytopathogenic Xanthomonas pathovars, with the ultimate aim of developing plant based formulations for plant disease management (Satish et al., 1999; Mohana et al., 2006; Kiran and Raveesha, 2006; Raghavendra et al., 2006). 318
Journal of Agricultural Technology Caesalpinia coriaria (Jacq.) Willd. distributed in tropical and subtropical region belongs to the family Caesalpinaceae is used in traditional medicine. Pods are used in the treatment of bleeding piles. This plant is good for emollient properties useful in treating freckles and alleviates acute colic pain (Anon, 2000). Considering these, a detail investigation was conducted to test the efficacy of the different solvent extracts against important phytopathogenic bacteria and to identify the bioactive compound responsible for the antibacterial activity. Materials and methods Collection of plant materials Fresh leaves and pods of Caesalpinia coriaria free from diseases were collected from Mysore (India), washed thoroughly 2-3 times with running tap water and once with sterile water, shade-dried, powdered and used for extraction. A voucher specimen of the plant is deposited in the herbarium of Department of Studies in Botany, University of Mysore, Mysore. Preparation of the aqueous extracts Fifty gm of shade dried, powder of leaves and pods of C. coriaria were macerated separately with 100 ml of sterile distilled water in a Waring blender (Waring International, new Hartford, CT, USA) for 10min. The macerate was first filtered through double layer muslin cloth and then centrifuged at 4000 g for 30 min. The supernatant was filtered through Whatman No. 1 filter paper and heat sterilized at 120ºC for 30 min. The extract was preserved aseptically in a brown bottle at 5ºC until further use. The extract was subjected to antibacterial activity assay. Preparation of solvent extractions Fifty gm of shade dried, powder of both leaf and pod of C. coriaria were filled separately in the thimble and extracted successively with 200 ml each of Petroleum ether, Benzene, Chloroform, Methanol and Ethanol using a Soxhlet extractor for 48 hours. All the extracts were concentrated using rotary flash evaporator. After complete solvent evaporation, each of these solvent extract was weighed and preserved at 5ºC in airtight bottles until further use. One gm of each solvent residue was dissolved in 10 ml of methanol which served as the test extracts for antibacterial activity assay.
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Phytochemical analysis of methanol extract Methanol extract that showed highest antibacterial activity was subjected to phytochemical analysis (Anon, 1985; Harborne, 1998) and active fraction separation such as Fraction I (Phenolic compounds), Fraction II (Neutral compounds), Fraction III (Bases) and Fraction IV (Weaker acids) following the procedures of Roberts et al. (1981). The active fraction was further resolved by TLC and column chromatography using silica gel G and H (Merck) respectively with mobile phases Chloroform : Acetone (1:1.5). All the corresponding fractions and spots were again subjected to antibacterial activity assay at 50 µl concentration. Plant pathogenic bacterial cultures Authentic pure cultures of phytopathogenic Xanthomonas axonopodis pv malvacearum (X. a. pv. m) isolated from cotton (Gossypium herbaceum L.), Xanthomonas axonopodis pv phaseoli (X. a. pv. p) isolated from french bean (Phaseolus vulgaris L.) and Xanthomonas campestris pv vasicatoria (X. c. pv. v) isolated from tomato (Lycopersicon esclentum mill.) were obtained from DANIDA lab, University of Mysore, India. Anti-bacterial activity assay Antibacterial activity of aqueous extract, solvent extracts and isolated constituents was determined by cup diffusion method on nutrient agar medium (Anon, 1996). Cups were made in nutrient agar plate using sterile cork borer (5 mm) and inoculum containing 106 CFU/ml of bacteria were spread on the solid plates with a sterile swab moistened with the bacterial suspension. Then 50 µl each of all aqueous, solvent extracts and isolated constituents were placed in the cups made in inoculated plates. The treatments also included 50 µl of sterilized distilled water and methanol separately which served as control. Antibiotics bacterimycin 2000 (Nitro propane hexadiol) (3 µg/ml) (Source: T. Stanes and Company Ltd., 23, Race-cource Road, Coimbatore-641018, India) and streptocycline (Streptomycin sulphate I.P. 90% Tetracycline Hydrochloride I.P. 10%) (1 µg/ml) (Source: Hindustan Antibiotics Ltd., PIMPRI, Pune-411018, India) at their respective recommended dosage were also treated for activity for comparative efficacy. The plates were incubated for 24 hours at 37ºC and zone of inhibition if any around the wells were measured in mm (millimeter). For each treatment six replicates were maintained. The data was subjected to statistical analysis using SPSS for windows software. The aqueous and methanol extracts of both leaf and pod showed highest 320
Journal of Agricultural Technology antibacterial activity, were further subjected to antibacterial activity assay at 10, 20, 30, 40, and 50 µl concentrations along with synthetic antibiotics bacterimycin and streptocycline for comparison. Results Anti-bacterial activity Aqueous extracts Anti-bacterial activity of aqueous leaf and pod extracts of Caesalpinia coriaria is presented in Table 1 and 2. Tukey HSD analysis of data revealed that, with increasing concentration of the aqueous extract, there was increase in antibacterial activity. Highly significant anti-bacterial activity of the aqueous extract at 50 µl was observed against all pathovars of Xanthomonas. Among the phytopathogenic Xanthomonas pathovars, Xanthomonas axonopodis pv. malvacearum was highly susceptible followed by Xanthomonas axonopodis pv. phaseoli and Xanthomonas campestris pv. vesicatoria. Pod extract recorded higher antibacterial activity than leaf extract. Solvent extracts The yield of extracts from leaf and pod were petroleum ether (1.8 & 1.1 gm), benzene (0.5 & 0.4 gm), chloroform (1.2 & 0.8 gm), methanol (26.8 & 31.0 gm) and ethanol (3.2 & 2.5 gm) respectively. Anti-bacterial activity of five different solvent extracts of both leaf and pod of Caesalpinia coriaria and synthetic antibiotics at 50µl concentration is presented in Table 1. Among the five solvents tested, methanol extract of both leaf and pod showed highly significant activity against all the test pathogens followed by ethanol and petroleum ether extract. Benzene and chloroform extracts of both leaf and pod did not show any activity against all Xanthomonas pathovars. The antibacterial activity of methanol extract of both leaf and pod of Caesalpinia coriaria at different concentrations is presented in (Table 2). Tukey HSD analysis of the data revealed that Xanthomonas axonopodis pv. malvacearum was highly susceptible among the Xanthomonas pathovars, where as Xanthomonas campestris pv. vesicatoria showed least inhibition.
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Table 1. Zone of Inhibitory activity (in millimeter) of different extracts of Caesalpinia coriaria and antibiotics against some plant pathogenic pathovars of Xanthomonas at 50 µl concentration.
1 2
Extracts Control aqueous Control methanol
3
Aqueous extract
4
Petroleum ether extract
5
Benzene extract
6
Chloroform extract
7
Methanol extract
8
Ethanol extract
9
Methanol extractPhenolic fraction
C C L P L P L P L P L P L P L P
X. a. pv.m 0.00 0.00
X. a. pv.p 0.00 0.00
X. c. pv.v 0.00 0.00
15.80±0.26 21.13±0.29 12.88±0.29 10.00±0.26 0.00 0.00 0.00 0.00
15.38±0.26 19.75±036 10.75±0.36 09.00±0.26 0.00 0.00 0.00 0.00
14.25±0.25 17.50±0.29 12.38±0.26 10.13±0.29 0.00 0.00 0.00 0.00
22.63±0.37 19.50±0.18 18.50±0.32 14.00±0.26 18.66±0.33
19.63±0.23 19.38±0.32 16.75±0.25 15.38±0.18 16.66±0.33
19.50±0.32 17.50±0.32 16.13±0.29 14.50±0.61 15.66±0.33
16.33±0.33
14.33±0.33
12.33±0.33
L 0.00 0.00 P 0.00 0.00 L 0.00 0.00 Methanol extract11 Neutral fraction P 0.00 0.00 L 14.33±0.33 14.00±0.57 Methanol extract12 Acidic fraction P 12.06±0.33 12.66±0.33 13 Streptocycline A 19.9±0.25 16.0±0.026 14 Bacterimycin 2000 A 10.00±0.43 11.38±0.026 Data given are mean of six replicates ± standard error, p < 0.0001 L- Leaf, P- Pod, C- Control, A-Antibiotic. X. a. pv.m - Xanthomonas axonopodis pv malvacearum X. a. pv.p - Xanthomonas axonopodis pv phaseoli X. c. pv.v - Xanthomonas campestris pv vasicatoria 10
322
Methanol extractbasic fraction
0.00 0.00 0.00 0.00 12.66±0.33 10.33±0.33 14.63±0.26 11.25±0.25
Journal of Agricultural Technology Table 2. Zone of Inhibitory activity (in millimeter) of aqueous and methanol extracts of Caesalpinia coriaria and antibiotics against some plant pathogenic pathovars of Xanthomonas at different concentrations. Organisms
Extracts
X .a. pv. m
Aq(L) Aq(P) Met(L) Met(P) Strept(A) Bact(A) Aq(L) Aq(P) Met(L) Met(P) Strept(A) Bact(A) Aq(L) Aq(P) Met(L) Met(P) Strept(A) Bact(A)
X. a. pv .p
X. c. pv. v
10µl 7.75±0.25a 9.50±0.18a 14.87±0.22a 10.25±0.25a 09.75±0.25a 00.00±0.00a 00.00±0.00a 9.00±0.26a 12.25±0.25a 12.88±0.29a 09.88±0.02a 00.00±0.00a 00.00±0.00a 9.75±0.25a 12.75±0.31a 10.13±0.29a 08.38±0.18a 00.00±0.00a
20µl 9.00±0.26b 14.00±0.26b 16.50±0.32b 13.63±0.37b 12.88±0.29b 07.00±0.26b 8.25±0.25b 14.88±0.29b 13.38±0.37b 13.38±0.12b 10.88±1.31b 06.88±0.02b 9.16±0.29b 11.88±0.29b 14.75±0.25b 12.75±0.25b 09.50±0.18b 07.13±0.29b
Concentrations 30µl 10.88±0.29c 18.00±0.26c 17.88±0.35c 14.63±0.26c 15.87±0.22c 08.88±0.35c 10.13±0.22c 16.13±0.29c 16.13±0.29c 16.25±0.36c 13.63±0.026c 08.13±0.022c 11.13±0.29c 14.38±0.37c 16.38±0.37c 15.00±0.26c 10.75±0.25c 08.50±0.32c
40µl 13.88±0.29d 19.75±0.25d 20.75±0.31d 16.13±0.29d 17.62±0.26d 10.00±0.26d 12.00d±0.26d 18.13±0.29d 17.50±0.23d 12.38±0.32d 14.75±0.02d 10.25±0.02d 14.00±0.26d 16.50±0.42d 12.75±0.31d 16.13±0.29d 12.63±0.26d 09.88±0.29d
50µl 15.00±0.26e 21.13±0.29e 22.50±0.32e 19.00±0.26e 19.95±0.25e 10.00±0.43e 15.38±0.26e 19.75±036e 19.25±0.25e 19.83±0.25e 16.00±0.26e 11.38±0.26e 14.25±0.25e 17.50±0.29e 19.38±0.26e 17.63±0.32e 14.63±0.26e 11.25±0.25e
Mean of six replicate ± standard error, The means followed by the same letter(s) are not significantly different at P< 0.05 when subjected to Tukey HSD (row by row comparisons). X. a. pv. m - Xanthomonas axonopodis pv malvacearum., X. a. pv. p - Xanthomonas axonopodis pv phaseoli., X. c. pv. v - Xanthomonas campestris pv vasicatoria. Aq- Aqueous, Met-Methanol, Strep- Streptocycline, Bact- Bacterimycin 2000, L- Leaf, P- Pod, A-Antibiotic.
Anti-bacterial activity of the methanol and ethanol extracts (leaf and pod) and aqueous extract (pod) was highly significant when compared with that of synthetic antibiotics Streptocycline and Bacterimycin 2000. Phytochemical analysis of methanol extract The phytochemical analysis of methanol extracts of both leaf and pod revealed the presence of carbohydrates and glycosides, protein and amino acid, phenolic compounds, saponin, tannin, flavonoids, oils, gum and mucilage. Further phytochemical analysis (Roberts et al., 1981) revealed that the antibacterial activity of methanol extract is due to the presence of phenolic and acidic compounds but the activity is less than that of crude methanol extract at 50 µl concentration (Table 1). In TLC separation phenolic fraction showed four 323
bands (Rf values 0.087, 0,333, 0.701, and 0.964) and acidic fraction showed five bands (Rf values 0.036, 0.079, 0.434, 0.565, and 0.876). Antibacterial activity was not observed in isolated compounds indicating the loss of antibacterial activity on further separation of the active fraction. Discussion The anti-bacterial activities of aqueous and solvent extracts were compared with standard Streptocycline and Bacterimycin 2000 and the results are reported in Table 1 and 2. The results show that the methanol extract of the plant parts showed more inhibitory effect than the other extracts. This tends to show that the active ingredients of the plant parts are better extracted with methanol than other solvents. The absence of antibacterial activity in the benzene and chloroform extracts indicates the insolubility of the active ingredients in these solvent. In general the activities against test bacterial culture used have shown good activity when compared with standard antibiotics. The phytochemical analysis of methanol extract revealed that the antibacterial activity is due to the presence of phenolic and acidic compounds and also observed that the activity is more in combination than separation. Further separation of the active fraction on TLC showed that the anti-bacterial activity was not observed in the isolated compounds. It is evident from the present investigations that the antibacterial activity in the methanol extracts of the leaf and pod but further separation of the methanol extract results in loss of antibacterial activity suggesting synergistic activity of the extract. There is a possibility of synergism between the compounds in a crude decoction than in isolated constituents (Daniel, 1999). Field existences of antibiotic resistant phytopathogenic bacteria are increasing in recent years (Mandavia et al., 1999). WHO banned many agriculturally important pesticides due to wide range of toxicity against nontarget organisms including humans which are known to cause pollution problem (Barnard et al., 1997). Some of the developing countries are still using these pesticides despite their harmful effects. Exploitation of naturally available chemicals from plants, which retards the reproduction of undesirable microorganism, would be a more realistic and ecologically sound method for plant protection and will have a prominent role in the development of future commercial pesticides (Verma and Dubey, 1999; Gottlieb et al., 2002). Many reports of antibacterial activity of plants extract against human pathogens and their pharmaceutical application are available (Cowan, 1999; Cragg et al., 1999; Newman et al., 2000; Gibbons, 2005), but not much has been done on the antibacterial activity of plants extract against plant pathogens (Satish et al., 1999). This is mainly due to lack of information on the screening/evaluation of 324
Journal of Agricultural Technology diverse plants for their antibacterial potential. Thus the present study reveals that C. coriaria is a potential candidate plant that could be successfully exploited for management of the diseases caused by different pathovars of Xanthomonas which are known to cause many diseases in wide variety of crops, causing considerable losses in yield and quality in an eco-friendly way. In the present investigations the antibacterial activity of C. coriaria against phytopathogenic bacteria has been demonstrated for the first time. Acknowledgements Authors are thankful to CSIR and AICTE New Delhi, for providing financial support.
References Agrios, G.N. (1997). Control of plant diseases. Plant pathology. 4th edition. California: Academic Press. Andrea, M.M., Peres, T.B., Luchini, L.C. and Pettinelli Jr., A. (2000). Impact of long-term pesticide applications on some soil biological parameters. Journal of Environmental Science & Health 35: 297-307. Anon. (1985). Pharmacopoeia of India. Government of India, New Delhi, Ministry of Health and family welfare. Anon. (1996). The Indian Pharmacopoeia. 3rd edition. Government of India, New Delhi. Ministry of Health and family welfare. Anon. (2000). The wealth of India, First supplement Series (raw material), Vol. 1. NISC and CSIR. New Delhi, India. pp.179. Balandrin, M.F., Klocke, J.A., Wurtele, E.S. and Bollinger, W.H. (1985). Natural plant chemicals: Sources of Industrial and Medicinal materials. Science 228: 1154-1160. Barnard, C., Padgitt, M. and Uri, N.D. (1997). Pesticide use and its measurement. International Pest Control 39: 161-164. Bender, C.L., Malvick, D.K., Conway, K.E., George, S. and Pratt, P. (1990). Characterization of pXV10A, a copper resistance plasmid in Xanthomonas campestris pv. vesicatoria. Applied and Environmental Microbiology 56: 170-175. Britto, S.J. and Senthilkumar, S. (2001). Antibacterial activity of Solanum incanum L. leaf extracts. Asian Journal of Microbial Biotechnology & Environmental Science 3: 65-66 Bylka, W., Szaufer-Hajdrych, M., Matalawska, I and Goslinka, O. (2004). Antimicrobial activity of isocytisoside and extracts of Aquilegia vulgaris L. Letters in Applied Microbiology 39: 93-97. Campos, A., Lino, C.M., Cardoso, S.M. and Silveira, M.I.N. (2005). Organochlorine pesticide residues in European sardine, horse mackerel and Atlantic mackerel from Portugal. Food Additives and Contaminants 22: 642-646. Chandler, J. (2005). Cost reduction in SIT programmes using exosect autodissemination as part of area wide integrated pest management. International Journal of Pest Control 47: 257-260. Clarke, J.H., Clark, W.S. and Hancock, M. (1997). Strategies for the prevention of development of pesticide resistance in the UK– Lessons for and from the use of Herbicides, Fungicides and Insecticides. Pesticides Science 51: 391-397. 325
Cowan, M.M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews 12: 564-582. Cragg, G.M., Boyd, M.R., Khanna, R., Kneller, R., Mays, T.D., Mazan, K.D., Newman, D.J. and Sausville, E.A. (1999). International collaboration in drug discovery and development: the NCl experience. Pure Applied Chemistry 71: 1619-1633. Daniel, M. (1999). Impediments preventing India becoming a herbal giant. Current Science 87: 275-276. Dekker, J. (1987). The risk for development of fungicide resistance a world wide problem. In Proceeding of the 11th International Congress of Plant Protection. October 5-7. Manila, Philippines. p. 318-321. Dorman, H.J.D. and Deans, S.G. (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Applied Microbiology 88: 308-316. Ferrer, A. and Cabral, R. (1991). Toxic epidemics caused by alimentary exposure to pesticides: a review. Food Additives and Contaminants 8: 755-776. Gassner, B., Wuthrich, A., Lis, J., Scholtysik, G. and Solioz, M. (1997). Topical application of synthetic Pyrethroids to cattle as a source of persistent environmental contamination. Environmental Science & Health 32: 729-739. Gibbons, S. (2005). Plants as a source of bacterial resistance modulators and anti-infective agents. Phytochemistry 4: 63-78. Gottlieb, O.R., Borin, M.R. and Brito, N.R. (2002). Integration of ethnobotany and phytochemistry: dream or reality?. Phytochemistry 60: 145-152. Harborne, J.B. (1998). Phytochemical methods: A guide to modern techniques of plant analysis. 3rd edition. Chapman & Hall Pub. London, UK. Harris, C.A., Renfrew, M.J. and Woolridge, M.W. (2001). Assessing the risks of pesticides residues to consumers: recent and future developments. Food Additives and Contaminant 18: 1124-1129. Hostettmann, K. and Wolfender, J. (1997). The search for biological active secondary metabolites. Pesticides Science 51: 471-482. Kapoor, A. (2001). Neem: The wonder plant. Pesticides Information 27: 33-34. Kilani, A.M. (2006). Antibacterial assessment of whole stem bark of Vitex doniana against some Enterobactriaceae. African Journal of Biotechnology 5: 958-959 Kiran, B. and Raveesha, K.A. (2006). Antifungal activity of seed extract of Psoralea corylifolia L. Plant Disease 20: 213-215. Mahajan, A. and Das, S. (2003). Plants and microbes- Potential source of pesticide for future use. Pesticides Information 28(4): 33-38. Mandavia, M.K., Gajera, H.P., Andharia, J.H., Khandar, R.R. and Parameshwaram, M. (1999). Cellwall degradation enzymes in host pathogen interaction of Fusarian wilt of chicken pea: Inhibitory effects of phenolic compounds. Indian Phytopathology 50: 548-551. Mathur, S.C. and Tannan, S.K. (1998). The pesticides industry in India. Pesticides information 24: 12-18. Mohana, D.C., Raveesha, K.A. and Lokanath Rai. 2006. Herbal remedies for the management of seed borne fungal pathogens by an edible plant Decalepis hamiltonii (Wight & Arn). Archives of Phytopathology and Plant Protection (in press ID no. jrmv55x6n267256r). Nafade, S.D. and Verma, J.P. (1985). Drug resistant mutants of Xanthomonas campestries pv malvacearum. Indian Phytopathology 38: 77-79. Newman, D.J., Cragg, G.M. and Snader, K.M. (2000). The influence of natural products upon drug discovery. Natural Products Research 17: 215-234.
326
Journal of Agricultural Technology Ortelli, D., Edder, P. and Corvi, C. (2005). Pesticide residues survey in citrus fruits. Food Additives and Contaminants 22: 423-428. Parameshwari, C.S. and Latha, T. (2001). Antibacterial activity of Ricinus communis leaf extracts. Indian drugs. 38: 587-588. Pimentel, D. and Levitan, L. (1986). Pesticides: Amounts applied and amounts reachingpests. BioScience 36: 86-91. Raghavendra, M.P., Satish, S. and Raveesha, K.A. (2006). Phytochemical analysis and antibacterial activity of Oxalis corniculata: a known medicinal plant. MycoScience 1: 72-78. Rath, C.C., Dash, S.K., Mishra, R.K. (2001). Invitro susceptibility of Japanese mint (Mentha arvensis L.) essential oil against five human pathogen. Indian Perfumer 45: 57-61. Roberts, R.M., Gilbert, J.C., Rodewald, L.B. and Wingrove, A.S. (1981). Modern experimental organic chemistry. The 3rdedition. Saunders golden sunburst series, Saunders college (Philadelphia), and Holt- Saunders Japan (Tokyo). pp 495-505. Rodriguez, H., Aguilar, L. and Lao, M. (1997). Variations in Xanthan production by antibioticresistant mutants of Xanthomonas campestris. Applied Microbiology and Biotechnology 48: 626-629. Saksena, S. (2001). Pesticides in farming: Enhancers of food security. Pesticide Information 27: 77 Satish, S., Raveesha, K.A. and Janardhana, G.R. (1999). Antibacterial activity of plant extracts on phytopathogenic Xanthomonas campestris pathovars. Letters of Applied Microbiology 8: 145-147. Shimpi, S.R. and Bendre, R.S. (2005). Stability and antibacterial activity of aqueous extracts of Ocimum canum leaves. Indian Perfumer 49(2): 225-229. Sundin, G. W., Demezas, D. H. and Bender, C. L. (1994). Genetic plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and Streptomycin bactericides. Applied and Environmental Microbiology 60: 4421-4431. Verma, J. and Dubey, N.K. (1999). Prospectives of botanical and microbial products as pesticides of Tomorrow. Current Science 76: 172-179. Verma, J.P., Dattamajumdar, S.K. and Singh, R.P. (1989). Antibiotic resistance mutant of Xanthomonas campestris pv malvacearum. Indian Phytopathology 42: 38-47. Weller, D.M. and Saettler, A.W. (1980). Colonization and distribution of Xanthomonas phaseoli and Xanthomonas phaseoli var. fuscans in field-grown Navy beans. Phytopathology 70: 500-506. Wheeler, W.B. (2002). Role of research and regulation in 50 years of pest management in agriculture. Agricultural Food Chemistry 50: 415-455. White, D.G., Zhao, S., Simjee, S., Wagner, D.D and McDermott, P.F. (2002). Antimicrobial resistance of foodborne pathogens. Microbes and Infection 4: 405-412. Williams, R. J. and Heymann, D. L. (1998). Containment of antibiotic resistance. Science 279: 1153-1154. Wodageneh, A. and Wulp, H.V.D. (1997). Obsolute pesticides in developing countries. Pesticides Information 23: 33-36.
(Received 9 September 2006; accepted 25 October 2006)
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